Neurotoxins produced by Clostridium botulinum are among the most potent toxins known to man. They are zinc endopeptidases that cleave a group of proteins integral to neurotransmitter release, causing flaccid paralysis, which may lead to death. The organism, and the toxin it produces, poses a significant threat as an agent of biological warfare and terrorism. Current therapy of botulism is based on macromolecular antitoxins, which are only able to neutralize the neurotoxins before they enter nerve cells, requiring treatment to commence as soon as possible to avoid permanent nerve damage. Consequently, there is a great need for cell-permeable small molecule drugs that selectively inhibit the action of botulinum neurotoxins. In combination with antibiotic therapy, such inhibitors could be life-saving. Of all seven serotypes produced by C. botulinum, we have chosen botulinum neurotoxin B (BoNT B) as the initial target for inhibitor development, since it is one of the most common forms and since it has been well-characterized structurally. In situ click chemistry, an exploratory new technique in which the target protein assembles its own inhibitors from simple precursors, will be employed for lead discovery and optimization. The main goals of the project are to (a) investigate the use of in situ click chemistry for the discovery and optimization of BoNT B inhibitors; (b) develop a detailed understanding of the structural requirements for BoNT B inhibition through structure-activity profiling, molecular modeling and X-ray crystallography; (c) develop compounds through in situ click chemistry that inhibit BoNT B with high affinity (<1 mu/M). The specific aims are as follows: (1) Search for anchor molecules: Produce and screen several libraries of peptidomimetic compounds that are targeted against the BoNT B metalloproteinase; (2) Perform target-guided synthesis of BoNT B inhibitors through in situ click chemistry; (3) Investigate the structure-activity relationship through in situ click chemistry and perform structure based studies.